Protein Profiling/Quantitative Proteomics
(Dean P. Edwards, Ph.D., Director;
Eastwood Leung, Ph.D., Co-Director; Shixia Huang, Ph.D., Co-Director)
The Dan L. Duncan Cancer Center is updating the services provided by "Protein Profiling/Quantitative Proteomics", as part of the existing Protein Expression/Proteomics Shared Resource led by Dr. Dean P. Edwards. These services are co-directed by Dr. Shixia Huang at Baylor College of Medicine and Dr. Eastwood Leung at Texas Children's Hospital and provides non-mass spectrometry (MS)-based platforms to measure the relative expression of multiple proteins in various biological samples. The following is a brief description of the services, technologies, prices and contact information.
Services at Baylor College of Medicine
Services at Texas Children's Hospital
Services at Baylor College of Medicine
Multiplex detection and quantitation of cytokines, phosphoproteins and other extra and intra-cellular signaling pathways.
- Technology & instrumentation: Bio-Plex suspension array system (Bio-Rad). This instrumentation is based on the Luminex xMAP bead technology. The principle is similar to a capture sandwich immunoassay (or ELISA) except the antibody to the target protein is covalently coupled to internal fluorescently dyed beads. The beads with bound target proteins are separated by laser excitation and quantitated. Beads coupled with different antibodies, each with a distinct fluoresence signature, are mixed thus enabling simultaneous assay of multiple protein targets in a single well of a 96 well plate. Assays can be done with small samples of cell and tissue extracts, cell culture media, serum and other biologic fluids. Bead kits are available for cytokines, phospho and total proteins of various signaling pathways and other regulatory proteins including transcription factors and markers of cancer, metabolism and autoimmunity. Enquire about availability of specific bead kits as they are continually changing and being developed commercially.
- Data Analysis: A Bio-Plex Manager Software station is available for assessment of data quality, data analysis, and quantitation.
Protein fractionation and/or differential protein expression profiling by 2 dimensional liquid chromatography
- Technology & instrumentation: ProteomeLab PF2D (Beckman Coulter). This is a high resolution 2 dimensional liquid protein fractionation system using chromatofocusing in the first dimension and a high capacity reverse phase HPLC column in the second phase. The system has an automated injection module for sample handling between first and second dimension columns, a sensitive UV detector and automated 96 well format robot for collection of samples from second dimension column. Selected liquid protein fractions can be further analyzed by mass spectrometry by the MS-based Proteomic Facility (Dr. Richard Cook). The PF2D system is designed to separate whole proteins from cell and tissue lysates and other biological fluids and is capable of separating as many proteins as traditional 2D SDS gel electrophoresis systems.
- Data analysis: ProteomeLab Software Suite is available for analysis of protein fractionation profiles.
Location: BCM Jewish Wing, Room 130D
Contact Information:
Shixia Huang, Ph.D.
Assistant Professor
Dan L. Duncan Cancer Center
Dept. of Molecular and Cellular Biology
Phone: 713-798-8722
E-mail: shixiah@bcm.edu
Prices
Bio-Plex:
- Cytokine (single plex) $ 487.75/96 well plate*
- Cytokines (10-plex)** $2157.25/96 well plate*
- Cytokines (20-plex)** $2897/96 well plate*
- Phospho proteins (single plex)**$ 827.00/96 well plate*
* The 96 well format will permit analysis of 70-80 test samples, the remaining wells are for standards and/or controls. For analyzing fewer samples, please enquire for details.
** Other combinations of multiplexing and types of beads are available. Please enquire for details. Price will vary dependent on the extent of multiplexing and the specific beads. Price includes technician time, equipment maintenance costs and consumable supplies.
PF2D Protein Fractionation:
| Service Type | Price/Sample |
|---|---|
| PF2D full run | $1550 |
| 1st dimensional run | $800 |
| 2nd dimensional run | $300 |
| 2nd dimension fraction pick/process | $20 |
| Double depletion (IgG/Albumin) Large scale | $222 |
| Double depletion (IgG/Albumin) Small scale | $119 |
| 1D SDS page | $40 |
| Coommassie blue stain | $20 |
| Silver stain | $40 |
Services at Texas Children's Hospital
The Genomics and Proteomics Core Laboratory (GPCL) of Texas Children's Hospital Cancer Centter operates as an integrated component under the network of the Baylor Dan L. Duncan Cancer Center. GPCL provides complementary protein profiling technology and subproteome purification services alongside the Protein Expression/Proteomics Shared Resource at Baylor. GPCL is directed by Dr. Eastwood Leung with the long term goal of biomarker discovery in complex biological samples. The laboratory provides two-dimensional gel electrophoresis-based protein profiling and subproteome purification such as glycoproteome or low abundance serum/plasma protein capturing. The following is a brief description of the services, technologies, prices and contact information.
Two-dimensional gel electrophoresis-based differential protein profiling
- Technology & instrumentation: Complex protein mixtures are separated by charge using isoelectric focusing in the first dimension. Further separation is achieved based on molecular mass of proteins using SDS-polyacrylamide gel electrophoresis in the second dimension. Over one thousand of protein spots can be resolved in optimized condition. Two gel formats are available: 1) The Zoom®IPGRunner? together with Novex gel from Invitrogen Inc consititute the minigel (8cm) format; 2) IPGphor II for the first dimension and Ettan DALTsix system for the second dimension from GE Healthcare Inc. constitute the large gel format. Protein gels can be stained with mass spectrometry compatible silver stain, sypro ruby, or Commassie blue stain. 2-D fluorescence differnece gel electrophoresis (DIGE) analysis will be available soon. Differential protein spots can be excised and processed for subsequent mass spectrometry analysis.
Subproteome purification
- Glycoproteome purification: Hydrazine-based solid phase extraction of glycoproteome is available. Depletion of abundance proteins in plasma or serum can be employed to enhance the discovery of low abunbance protein markers in body fluid. Captured subproteome will be subjected to gel electrophoresis-based separation.
Location: Feigin Center (1102 Bates Street, Houston, TX77030) Room C1070.01
Director:
Eastwood Leung Ph.D.
Assistant Professor
Dan L. Duncan Cancer Center
Dept. of Pediatrics and Dept of Cellular and Molecular Biology
Phone: 832-824-4373
E-mail: exleung@texaschildrenshospitals.org
Personnel:
Matthew Folsom B.A., CLDir, CLSp (CG)
Laboratory manager
Office: 832-824-4458
Email: mrfolsom@texaschildrenshospital.org
Sergery Torsky, Ph.D.
Bioinformatics programmer
Phone: 832-824-1036
Sivashankarappa Gurusiddappa
Staff Technologist
Phone: 832-824-4453
Main Laboratory:
Phone: 832-824-4354
Fax: 832-825-4038
Prices
| Services | Price/Sample |
|---|---|
| Isoelectric focusing small gel format | $25 |
| Isoelectric focusing large gel format | $30 |
| 2nd dimension SDS-PAGE small gel format | $40 |
| 2nd dimension SDS-PAGE large gel format | $60 |
| Commassie blue stain of small gel | $10 |
| Commassie blue stain of large gel | $30 |
| Sypro ruby stain of small gel | $30 |
| Sypro ruby stain of large gel | $80 |
| Mass spectrometry-compatible silver stain of small gel | $25 |
| Mass spectrometry-compatible silver stain of large gel | $55 |
| 2D gel image analysis | $25 |
| Hydrazine-based glycoprotein capture assay | $75 |
More information of the GPCL in the Texas Children's Hospital